Method for the detection of cancer

ABSTRACT

The present invention relates to a method for the diagnosis and/or the follow up of the evolution of cancer, which includes the analysis and quantification of over expressed and amplified genes in the plasma/serum of cancer patients or persons suspected to harbor cancer. This is achieved by analyzing together the amount of DNA and RNA of certain genes in the plasma/serum of cancer patients that are the reflection of a gene amplification and/or a gene over expression in comparison to healthy controls.

Notice: More than one reissue application has been filed for the reissueof U.S. Pat. No. 7,700,286, which claims the benefit of European PatentApplication No. 05007508, which was filed on Apr. 6, 2005. Thisapplication is a reissue divisional of U.S. patent application Ser. No.13,447,104, entitled “Methods for Detection of Cancer,” filed on Apr.13, 2012, which is an application for reissue of U.S. Pat. No.7,700,286. The reissue applications are the present reissue divisionalapplication and the parent reissue application Ser. No., 13/477,104,which issued as RE 44,596. The instant application adds new claimsrelative to the original patent, U.S. Pat. No. 7,700,286. The entirecontents and disclosures of the above applications are herebyincorporated by reference.

The present invention describes a method of diagnosis and/or follow upof the evolution of most types of cancer, for instance after achemotherapy or after an operation.

It is known that diagnosis and follow up of the evolution of cancer aredone, besides direct observation of the tumors, by biopsy analysis or inthe case of blood malignancies by analysis of the bone marrow. Thisimplies either a surgical intervention or an invasive test such as abiopsy or a bone marrow aspiration. Now, without taking into account thedisagreeable or even dangerous aspect of such methods it has beenobserved that they could moreover not be very precise.

Conventional methods of diagnosis are not very satisfactory. As anexample, colorectal cancer screening presently relies on fecal occultblood testing (FOBT) which is both insensitive and non-specific. Incontrast, flexible sigmoidos-copy is sensitive and specific for earlydistal disease but is both invasive and insensitive for proximaldisease. Furthermore, barium enema is relatively sensitive and specificbut requires colonic preparation, radiation and a day off work, whiletotal colonoscopy is highly sensitive and specific but is also invasiveand expensive.

The situation appears little better for other cancers. No reliable testis available for early detection of lung cancer, with computerizedtomography being the most reliable tool.

An important strategy to reduce mortality from breast cancer is theintroduction of mammographic screening in an attempt to detect cancersat an asymptomatic and pathologically early stage. Although severalstudies indicate that mass screening is a useful strategy for reducingbreast cancer mortality, there are a number of disadvantages associatedwith this form of cancer screening. These include a high rate of falsepositive tests, frequent false negative tests and the enormous publichealth costs involved. Thus, when the benefits of mammographic screeningare weighed against its costs and other disadvantages, it is perhaps notsurprising that this form of screening has engendered an enthusiasticand contentious debate over the past 20 years.

Finally, development of conventional protein tumor markers, such ascarcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), along withthe widely used prostate specific antigen (PSA) was driven largely bythe introduction of new methods for quantifying small amounts ofcirculating proteins. However, sensitivity and specificity shortcomingswith these assays remain to be overcome.

The aim of this invention consists therefore in providing a method ofdiagnosis and/or follow up of the evolution of most types of cancerwhich would be, on one hand, more precise and trustworthy and, on theother hand easier to perform without implying an invasive test for thepatient.

Small amounts of free DNA circulate in both healthy and diseased humanplasma or serum, and increased concentrations of plasma or serum DNA arepresent in cancer patients. The present inventors were the first todemonstrate that this DNA extracted from the plasma of cancer patientshas tumor related characteristics. They include decreased strandstability, oncogene and tumor suppressor gene mutations, micro-satellitealterations, and gene hypermethylation. This has led to suggest that anon-invasive diagnostic test for cancer might be feasible using thesemolecular techniques.

Using essentially similar molecular techniques, tumor related mRNA havebeen detected in the plasma of cancer patients. These RNA markers arethe result of an over expression of some genes in the cancer cells andmay be found in increased quantities in the plasma/serum of cancerpatients compared to healthy controls.

Now this over expression of genes is often accompanied by anamplification of the same gene in the cancer cells, and the presentinventors have found that this amplification can be seen subsequently inthe plasma/serum of the patient. It should be stressed that thisamplification is independent of the fact that there is, as mentionedabove, usually more plasma/serum DNA in cancer patients than in healthycontrols.

The present inventors have therefore developed a cancer detection assayin plasma/serum measuring by adding and comparing the amount of DNA andRNA of certain genes in the plasma/serum of cancer patients that are thereflection of a gene amplification and a gene over expression. Thus geneamplification (seen by more DNA) and gene over expression (more RNA) arelinked.

Consequently, the object of the present invention, reaching theabove-mentioned aim, is consisting of a method for the diagnosis or thefollow up of the evolution of cancers which comprises measuring togethergene over expression (RNA) and gene amplification (DNA) in the bodilyfluids of patients suspected to harbor cancer on any gene that is bothamplified and over expressed in cancer cells and comparing to healthycontrols.

More particularly, RNA and DNA are extracted from a bodily fluid, suchas plasma, serum, sputum, saliva, etc, purified and amplified, and theover expressed RNA and amplified DNA are analyzed and compared to aunique house keeping gene.

As examples, the genes analyzed can be selected from hTERT, hTR, TEP1,MYCN, MYCC, ErbB2, Her2, Her2/Neu, Her1, Cyclin A and D1, ABL, SKP2,ETV6 (TELgene), MGC2177, PLAG1, PSMC6P and LYN.

Preferably, the nucleic acids are amplified by reversed transcriptasechain reaction (RT-PCR) and are analyzed by gel coloration, byradioactive immunological technique (RIA), by enzyme linkedimmunosorbant test (ELISA) or by a microchip test (gene array), andpossibly quantified by any method for nucleic acid quantification.

The quantification of RNA and DNA can advantageously be carried out byreal time PCR, such as “TAQMAN™”, or on capillaries “LIGHTCYCLER™”, orreal time PCR and RT PCR of any company.

Furthermore, the genes analyzed may be compared to a reference nucleicacid extract (DNA and RNA) corresponding to the expression (RNA) andquantity (DNA) of a unique house keeping gene, or to a reference RNAcorresponding to the expression of a house keeping coding gene, or to areference DNA corresponding to a unique gene, or may be estimated inreference to a standard curve obtained with nucleic acids of a cellline.

In the following description of the present invention, telomerase RNAand DNA have been chosen as example, since telomerase activity isenhanced in 85 to 100% of cancers. But it must be stressed that thepresent invention is valid for all genes, and specially oncogenes thathave been reported to be both amplified and over expressed in manycancers and cancer cell lines, for instance MYCN in neuroblastoma, ErbB2in esophagal, breast and ovarian cancer, Her2, Her2/Neu and Her1 inbreast and Her2/Neu in lung, Cyclin A an D1 in colorectal or laryngealcancer, ABL in leukemias and lymphomas, SKP2 in non small cell lungcancer, ETV& (TEL-gene) in myelodysplastic syndrome. These are some ofthe most studied, but many others have been reported, such as MGC2177,PLAG1, PSMC6P, and LYN.

To illustrate the present invention, the hTERT gene was used, whichcodes for the reverse transcriptase of the telomerase ribonucleoprotein.

Telomerase is a ribonucleoprotein enzyme that synthesizes repeatedtelomeric sequences at chromosomal ends. The telomeres protect thechromosomal ends and at each cell division these telomeres areshortened. Telomerase composed of an RNA template (hTR) and a reversetranscriptase enzyme (hTERT) plus associated proteins such as TEP 1 thatsynthesizes these telomeres.

The activity of this enzyme has become an accepted indicator for thediagnosis and the prognosis of most malignant tumors. The expression ofhuman telomerase RNA (hTR) or of the reverse transcriptase enzyme of theRNA telomerase (hTERT) or of the associated protein (TEP1) has beenmeasured during the progression of several types of tumors. This hasenabled the establishment of a correlation between this expression (theamount of RNA) and telomerase activity. Most cancers and immortalizedcell lines have a high telomerase activity that reflects a mechanismthat escapes normal aging regulations. We have a patent in Europe and apending patent in the US for the measurement of the amount of mRNA inthe plasma/serum coding for hTERT and TEP1.

Now, an amplification of the genes (DNA) coding for telomerase subunits(especially hTERT) has been observed in cancer cell lines and indifferent kinds of cancers. The present inventors have further observedan amplification of the hTERT gene in the plasma of cancer patients.

Although RNA components and mRNA coding for telomerase are cellularcomponents, it was observed that, surprisingly, these components couldbe also found in an extracellular form in plasma or serum.

Indeed when both nucleic acids are extracted and amplified, thedifference between the healthy controls and the cancer patients issurprisingly higher.

To sum up the method, the present inventors have shown an increasedamount of hTR, hTERT and TEP1 RNA in the plasma or serum of personssuffering from breast, ovarian, head and neck, pancreatic, liver,stomach or colon cancer while these products have been shown to beabsent in the blood of healthy persons. Moreover DNA coding fortelomerase components in particular for hTERT can also be found ingreater amounts (amplified) in the plasma of cancer patients than inhealthy controls. It is known since a long time that there is often moreDNA in the plasma of cancer patients than in the plasma of healthycontrols (this has been demonstrated by measuring the amount ofbeta-globin for instance), but hTERT DNA yields even more than whatcould be expected, giving evidence of an amplification of this gene.

More precisely, the method of diagnosis according to the inventionconsists in extracting the nucleic acids (RNA and DNA) from the plasmaor the serum of the blood, purifying it and amplifying it in order toestablish the presence and the quantity of the product made in this caseby the of components hTERT. This shall be done in a comparative mannerbetween the plasma or serum of a person suspected of malignancy and theplasma or serum of a healthy person or of a control suffering from anon-malignant disease.

The amplification product of the DNA and of the RNA componentstranscribed into DNA by RT-PCR are detected and quantified. This can bedone by any nucleic acid quantification method.

Similarly, any technique of extraction of purification and ofamplification of the nucleic acids (DNA and RNA) in the plasma or theserum may be used.

The present invention will now be illustrated in a non-limitative mannerby the following example related to the diagnosis of some cancers usinghTERT DNA and RNA quantification.

EXAMPLE

Diagnosis of different cancers by the detection of amplified hTERT DNAand over exypressed hTERT RNA in the plasma or serum of the blood.

Blood samples (2 ml) were collected in EDTA tubes prior to surgery ortreatment on patients bearing small malignant breast tumors or onpatients suffering of head and neck, colorectal, pancreatic and livercancer. Blood was taken in the same way as healthy volunteers forcontrols.

To guarantee good quality plasma nucleic acids, the whole blood samplesshould be centrifuged as soon as possible. If the centrifugation cannottake place immediately, the blood samples should be stored at 4°immediately after blood collection and centrifuged within 6 hours. Theblood samples at 1,600 g for 10 min at 4° C. The plasma was transferredinto new tubes taking care not to disturb the buffy coat layer. A secondround centrifugation of the plasma was performed at 16,000 g for 10 minat 4° C. The plasma was finally transferred into new tubes taking carenot to disturb the underlying cell pellet and stored if necessary at−70°.

RNA and DNA were extracted using a commercially available kit (Ultrasensviral kit from Qiagen), which extracts DNA as well as RNA, according tomanufacturers instructions.

The primers and TAQMAN™ probe for hTERT were located on one exon andwhich would yield both RNA and DNA:

F: 5′-ACC GTC TGC GTG AGG AGA TC-3′; (SEQ ID NO: 1)R: 5′-CCG GTA GAA AAA AGA GCC TGT TC-3′ (SEQ ID NO: 2) and the PROBE5′Fam -TGT ACG TCG TCG AGC TGC TCA GGT CTT T-3′ TAMRA (SEQ ID NO: 3).

As reference for RNA and DNA we used the beta-Globin gene on exon 2:forward primer: 5′ CTGCTGGTGGTCTACCCTTG 3′ (SEQ ID NO: 4); Reverseprimer: 5′ CCTGAAGTTCTCAGGATCCA 3′ (SEQ ID NO: 5); and Hybridizationprobe:5′Fam. CTCCTGATGCTGTTATGGGCAACCCT 3 TAMRA′ (SEQ ID NO: 6) whichwould yield both RNA and DNA or the GAPDH gene on exon 8: Forward primer5′GTGGACCTGACCTGCCG3′ (SEQ ID NO: 7); Reverse primer 5′GGAGGAGTGGGTGTCGC 3′ (SEQ ID NO: 8) and the probe for TAQMAN™ 5′FAM-AAGGGCATCCTGGGCTACACTGAGCA3′ TAMRA (SEQ ID NO: 9).

These reference primers for RNA and DNA can be replaced by anyhousekeeping unique gene. The results given below were calculated usingarbitrary quantities expressed either as CT (cycle threshold numbers) or2 ^(ΔCT) values (for instance 2 ^(CT) of hTERT^(−CT) of b-Globin). Theyalways compared extractions of plasma nucleic acids of cancer patientsand healthy donors extracted the same day and with the same amount (0.5ml) of plasma/serum. It is possible to estimate in another way bycomparing the results to a curve obtained by known quantities of onegene.

The QuantiTect Probe RT-PCR (Qiagen) was used in 25 μl RT-PCR reactionmixture containing the manufacturer's Master Mix, the RT mix(Omniscript™ reverse transcriptase, Sensiscript™ reverse transcriptase,hot-start Taq™ DNA polymerase) to which we added the set of primers (0.4μM) and TAQMAN™ probe (0.1 μM) and 3 to 6 μl of the 30 μl of elutednucleic acids. The RT-PCT conditions of the mixture were an initialincubation at 50° C. for 30 min followed by a 95° C. incubation for 15min to activate the HotstarTaq™ DNA Polymerase, then 50 cycles at 94° C.(15 sec), 60° C. (1 min).

All base sequences mentioned here above as primer examples are known andmay as such be consulted on the web site of the Genome Database. Theymay be replaced by other primers and probes located on theabove-mentioned genes. Reference genes may be changed by other genes.

Results Obtained:

Data have been obtained on 74 cancer patients and 51 controls with 98%specificity. The sensitivity changes from cancer to cancer ranging from81% to over 90%. The cancer patients suffered from head and neck,breast, colorectal, pancreatic and liver cancers.

The results obtained by Real Time Quantitative RT PCR measuring both DNAand RNA of hTERT compared to beta-Globin gene in the plasma of cancerpatients and healthy controls are presented on the following Table.

Samples Number of hTERT studied samples positive % CONTROLS 51  2%PANCREATIC 27  81% CANCER HEAD AND 16  94% NECK COLORECTAL 18  83%BREAST 7 100% LIVER 6  83%

Furthermore, the results obtained are illustrated on the annexedfigures, where;

FIG. 1 shows as reference the amplification plots obtained using realtime quantitative PCR for the hTERT gene (DNA), and with the x-axisbeing the cycle number of the PCR reaction and the y-axis thefluorescence intensity over background.

FIG. 2 shows as reference the amplification plots obtained using realtime quantitative RT-PCR for the hTERT gene (RNA), and with the x-axisbeing the cycle number of the PCR reaction and the y-axis thefluorescence intensity over background.

FIG. 3 shows the amplification plots obtained using real timequantitative RT-PCR for the hTERT gene (total nucleic acids DNA andRNA), according to the present invention, and with the x-axis being thecycle number of the PCR reaction and the y-axis the fluorescenceintensity over background.

As it can easily be seen on FIG. 1 , the first group of lines (A) with aCT value around 37 is composed of the amplification product of samplesof DNA from healthy donors with hTERT primers, and the second group (B)with a CT value around 35 is composed of the amplification product ofsamples of plasma DNA from patients suffering from head and neck cancer.

On FIG. 2 , the first group of lines (C) with a CT value around 34 iscomposed of the amplification product of samples of RNA from healthydonors with hTERT primers, and the second group (D) with a CT valuearound 32 is composed of the amplification product of samples of plasmaRNA from patients suffering from head and neck cancer. A difference of 2CT values represents a difference of 4 times RNA values obtained fromthe same amount of plasma.

On FIG. 3 , which represents the results the first group of lines (E)with a CT value around 35 is composed of the amplification product ofsamples of plasma nucleic acid from healthy donors with hTERT primers,and the second group (F) with a CT value around 29 is composed of theamplification product of samples of plasma nucleic acid from patientssuffering from head and neck cancer. The difference between the CTvalues (comprising RNA and DNA) of the control group and the cancergroup is higher than in FIGS. 1 and 2 , DNA or RNA are measured. Thisdemonstrates the clear advantage of the method according to the presentinvention.

The invention claimed is:
 1. A method for the diagnosis or the follow upof the evolution of cancers which comprises measuring both geneover-expression (RNA) and gene amplification (DNA) of a gene present inthe bodily fluids of a patient that is both amplified and over-expressedin cancer cells and comparing to healthy controls.
 2. The methodaccording to claim 1, wherein RNA and DNA are extracted from a bodilyfluid, purified and amplified, and the over-expressed RNA and amplifiedDNA are analyzed and compared to a house keeping gene.
 3. The methodaccording to claim 2, wherein the genes analyzed are selected fromhTERT, hTR, TEP1, MYCN, MYCC, ErbB2, Her2, Her2/Neu, Her 1, Cyclin A andD1, ABL, SKP2, ETV6 (TELgene), MGC2177, PLAG1, PSMC6P and LYN.
 4. Themethod according to claim 2, wherein the nucleic acids are amplified byreverse transcriptase chain reaction (RT-PCR).
 5. The method accordingto claim 3, wherein the nucleic acids are amplified by reversetranscriptase chain reaction (RT-PCR).
 6. The method according to claim1, wherein the genes analyzed are selected from the group consisting ofhTERT, hTR, TEP1, MYCN, MYCC, ErbB2, Her2, Her2/Neu, Her 1, Cyclin A andD1, ABL, SKP2, ETV6 (TELgene), MGC2177, PLAG1, PSMC6P and LYN.
 7. Themethod according to claim 6, wherein the nucleic acids are amplified byreverse transcriptase chain reaction (RT-PCR).
 8. The method accordingto claim 1, wherein the nucleic acids are amplified by reversetranscriptase chain reaction (RT-PCR).
 9. The method according to claim1, wherein the genes analyzed are compared to a reference nucleic acidextract (DNA and RNA) corresponding to the expression (RNA) and quantity(DNA) of a house keeping gene.
 10. The method according to claim 1,wherein the genes analyzed are compared to a reference RNA correspondingto the expression of a house keeping gene.
 11. The method according toclaim 1, wherein the genes analyzed are compared to a reference DNAcorresponding to a housekeeping gene.
 12. The method according to claim1, wherein the gene quantification may be estimated in reference to astandard curve obtained with nucleic acids of a cell line.
 13. Themethod according to claim 1, wherein the nucleic acids RNA and DNA areanalyzed by gel coloration, by radioactive immunological technique(RIA), by enzyme linked immunosorbant test (ELISA) or by a microchiptest (gene array), and quantified by any method for nucleic acidquantification.
 14. The method according to claim 1, wherein the nucleicacids RNA and DNA are quantified by real time RT PCR.
 15. The methodaccording to claim 1, wherein the RNA and DNA are extracted from thepatient and measured simultaneously.
 16. A method for measuring bothgene over-expression (RNA) and gene amplification (DNA) in a patientsuspected of having cancer, wherein the gene is selected from the groupconsisting of hTERT, hTR, TEP1, MYCN, MYCC, ErbB2, Her2, Her2/Neu, Her1, Cyclin A, Cyclin D1, ABL, SKP2, ETV6 (TELgene), MGC2177, PLAG1,PSMC6P and LYN, comprising: obtaining a sample of a bodily fluid from apatient, and extracting both RNA and DNA simultaneously from saidsample, measuring both gene over-expression (RNA) and gene amplification(DNA) at the same time in said sample, and comparing said measurement toa healthy control.
 17. The method according to claim 16, wherein RNA andDNA are extracted from a bodily fluid, purified and amplified, and theover-expressed RNA and amplified DNA are analyzed and compared to ahouse keeping gene.
 18. The method according to claim 17, wherein theover-expressed and amplified gene is hTERT.
 19. The method according toclaim 18, wherein the nucleic acids are amplified by reversetranscriptase chain reaction (RT-PCR).
 20. The method according to claim16, wherein the genes analyzed are compared to a reference nucleic acidextract (DNA and RNA) corresponding to the expression (RNA) and quantity(DNA) of a house keeping gene.
 21. The method according to claim 16,wherein the genes analyzed are compared to a reference RNA correspondingto the expression of a house keeping gene.
 22. A method for detectinggene amplification of one or more genes that occurs in cancer cells of asubject having cancer, the gene amplification of the one or more genesbeing indicative of cancer, the method comprising: (i) obtaining a bloodsample from the subject; (ii) separating cell-free deoxyribonucleic acid(DNA) from a plasma or serum fraction of the blood sample; (iii)amplifying DNA of the one or more genes of the subject present in thecell-free DNA separated from the plasma or serum fraction; (iv)amplifying DNA of a reference gene present in the cell-free DNAseparated from the plasma or the serum fraction; (v) measuring amountsof the amplified DNA of the one or more genes; (vi) measuring an amountof the amplified DNA of the reference gene, wherein the reference geneis different than the one or more genes; (vii) determining if theamounts of the amplified DNA of the one or more genes are increasedrelative to the amount of amplified DNA of the reference gene; and(viii) detecting the gene amplification in cancer cells of the one ormore genes if the amounts of the amplified DNA of the one or more genesare determined to be increased relative to the amount of amplified DNAof the reference gene.
 23. The method according to claim 22, wherein theone or more genes are a plurality of genes.
 24. The method according toclaim 22, wherein the one or more genes include oncogenes.
 25. Themethod according to claim 22, further comprising detecting atumor-related characteristic in the one or more genes present in theplasma or serum fraction.
 26. The method according to claim 22, whereinthe separating comprises extracting cell-free DNA from the plasma orserum fraction.
 27. The method according to claim 26, further comprisingamplifying DNA of the one or more genes of the extracted cell-free DNA.28. The method according to claim 27, wherein the amplifying comprisesusing polymerase chain reaction.
 29. The method according to claim 22,wherein the one or more genes are selected from the group consisting ofhTERT, hTR, TEPI, MYCN, MYCC, ErbB2, Her2, Her2/Neu, Her1, Cyclin A andD1, ABL, SKP2, ETV6 (TELgene), MGC2177, PLAG!, PSMC6P and LYN.
 30. Themethod according to claim 22, wherein the cancer is neuroblastoma, andwherein the one or more genes include MYCN.
 31. The method according toclaim 22, wherein the cancer is selected from the group consisting ofesophageal cancer, breast cancer, and ovarian cancer, and wherein theone or more genes include ErbB2.
 32. The method according to claim 22,wherein the cancer is breast cancer or lung cancer, and wherein the oneor more genes are selected from the group consisting of: Her2, Her2/Neu,and Her1.
 33. The method according to claim 22, wherein the cancer iscolorectal cancer or laryngeal cancer, and wherein the one or more genesinclude Cyclin A or Cyclin D1.
 34. The method according to claim 22,wherein the cancer is selected from the group consisting of: leukemiaand lymphoma, and wherein the one or more genes include ABL.
 35. Themethod according to claim 22, wherein the cancer is non-small cell lungcancer, and wherein the one or more genes include SKP2.
 36. The methodaccording to claim 22, wherein the cancer is myelodysplastic syndrome,and wherein the one or more genes include ETV6.
 37. The method accordingto claim 22, wherein the cancer is selected from the group consisting ofbreast cancer, ovarian cancer, head and neck cancer, pancreatic cancer,liver cancer, stomach cancer, and colorectal cancer, and wherein the oneor more genes include hTR, hTERT, or TEPI.
 38. The method according toclaim 22, wherein the cancer is nasopharyngeal cancer, and wherein theone or more genes is not Epstein Barr Virus DNA.
 39. The methodaccording to claim 22, further comprising following up with the subjectbased on the presence of the gene amplification.